FPN is transiently present on phagosomes containing S aureus. (A) Confocal images of RAW macrophages expressing FPN-GFP (in green) having been exposed to live eFluor-670–labeled S aureus (in blue) for 5 and 15 minutes are shown. Bacteria that were extracellular at 5 minutes were marked with a TRITC-conjugated secondary antibody (in red). Arrows point to phagocytosed S aureus that are demarcated by GFP fluorescence at 5 minutes. In the bottom panels, the arrowheads point to phagocytosed S aureus that are not demarcated by GFP at 15 minutes. The white arrow points to an S aureus coccus that is enveloped by GFP at the same time point; however, the presence of TRITC fluorescence indicates this bacterium was engulfed after the 5- minute time point. The dashed lines indicate the areas of the cell analyzed by the line scans that are presented to right of the micrographs for 5 minutes (top graph) and 15 minutes (bottom graph). Scale bars, 10 μm. (B) The localization of FPN-GFP to phagosomes containing S aureus expressing mCherry (top row, in red), IgG-opsonized latex beads (middle row), and S typhimurium expressing RFP (bottom row, in red) at 1 hour postphagocytosis is shown. IgG-opsonized beads remaining extracellular at 1 hour were marked with an anti-human Cy3-conjugated antibody and are in red (middle panel). Cells were also immunostained for endogenous LAMP-1 (in blue). Scale bars, 10 μm. (C) The fraction of FPN-GFP–positive phagosomes at 1 hour postphagocytosis is plotted for the 3 distinct phagocytic targets shown in panel B. These data are the mean ± SD from 3 independent experiments. Statistical significance was determined using an ordinary 1-way analysis of variance and a Tukey multiple comparison. n.s., not significant. (A-B) The images were acquired from fixed samples at the indicated times using laser scanning confocal microscopy. *Indicates the position of representative bead containing phagosomes. p.i., postinfection.